1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and high purity of the test material the test concentrations used in the definitive test were prepared by diluting (with culture medium) a saturated solution prepared from initial test material dispersions at a concentration of 150 mg/L.
In each of four group glass stoppered conical flasks an amount of test material (75 mg) was dispersed in culture medium (500 mL) to give initial test material dispersions at a concentration of 150 mg/L. The test material dispersions were shaken at 300 rpm at a temperature of 30 degC for a period of 48 hours. After the shaking period the contents of the replicate flasks were pooled and the undissolved test material removed by filtration (0.2 micron, first approximate 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 11 mg/L from with a series of dilutions were made to give 5.5, 2.75, 1.38 and 0.69 mg/L stock solutions.
Media preparation trials conducted using reverse osmosis purified water as the diluent indicated that when using a similar method of preparation as described above, a measured concentration (based on Dissolved Organic Carbon analysis) of a similar magnitude to the results of the water solubility test was obtained. It was therefore considered that filtration was an appropriate method for the removal of undissolved test material.
An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 0.69, 1.38, 2.75, 5.5 and 11 mg/L.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Institute of Freshwater Ecology, United Kingdom. Cultures were maintained in the laboratory by the periodic peplenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 +/- 1 degC under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 +/- 1 degC throughout the test.

pH:

The test material vessels showed an increase in pH over the 72 hour test period following a concentration dependant pattern with the lower test material concentrations exhibiting a great increase in pH. This effect was considered to be due to there being greater numbers of viable cells in the lower test concentrations and hence greater utilisation of carbonates and bicarbonates from photosynthesis/respiration.

Nominal and measured concentrations:

Nominal: 0.69, 1.38, 2.75, 5.5 and 11 mg/l.

Details on test conditions:

Media Preparation Trials:
Media preparation trials were conducted in order to determine the most suitable method obtaining a saturated solution at a concentration similar to the water solubility level of 32 mg/L as determined by 4.8 Water Solubility Safepharm 2003.

- Method 1
An amount of test material (1650 mg) was dispersed in 11 liters of reverse osmosis purified deionized water with the aid of propeller stirring at approximately 2000 rpm at a temperature of 21 degC for 48 hours to give an initial test material dispersion of 150 mg/L. After 48 hours the stirring was stopped and the undissolved test material was removed by filtration (0.2 micron) to give a saturated solution. Samples were taken from this saturated solution to determine the amount of dissolved test material by Dissolved Organic Carbon (DOC) analysis. The amount of test material in solution was shown to be 23 mg/L.

- Method 2
An amount of test material (75 mg) was dispersed in 500 ml of reverse osmosis purified deionized water with the aid of shaking at approximately 300 rpm at a temperature of 30 degC for 48 hours. After 48 hours the shaking was stopped and the undissolved test material was removed by filtration (0.2 microm filter) to give a saturated solution. Samples were taken from this saturated solution to determine the amount of dissolved test material by Dissolved Organic Carbon (DOC) analysis, The amount of test material in solution was shown to be 21 mg/L.

Range Finding Test:
The results of the media preparation trials showed that the dissolved test material concentration obtained from both Methods 1 and 2 was slightly lower than the water solubility level of the test material (32 mg/L). This was considered possibly to be due to the filters used in the media preparation trials not being pre-conditioned by passing a suitable volume through the filter prior to taking the sample for DOC analysis. However, given that the results of the DOC analyses were of a similar magnitude to the results of the water solubility test, it was considered that filtration was appropriate for removal of the undissolved test material if pre-conditioning of the filters was conducted.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. For the purpose of the range-finding test the shake-flask method (Method 2) was used for the preparation of a saturated solution of the test material. The test was conducted in 250 mL glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was prepared as a saturated solution from initial test material dispersions of 150 ml/L.
In each of four ground glasses stoppered conical flasks amount of test material (75 mg) were dispersed in culture medium (500 mL) to give initial test6 material dispersions at a concentration 150 mg/L. The test material dispersions were shaken at 300 rpm at a temperature of 30 degC for a period of 48 hours. After the shaking period the contents of the replicate flasks were pooled and any undissolved test material was removed by filtration (0.2 micron filter, first approximate 1 liter discarded to pre-condition the filter) to produce a saturated solution.
A saturated solution prepared in an identical manner to that used for the range-finding test to provide samples for preliminary recovery and stability analyses showed that the dissolved test material concentration of 21 mg/L determined by DOC analysis during the media preparation trials when reverse osmosis purified deionized water was used as the diluent.
Examination of the test material structure indicated that it was a zinc salt with the anion being a weak acid. It was therefore considered that the weak acid may associate with other cations of limited solubility leading to precipitation. As a result of this the maximum solubility of the test material in culture medium was reduced by the presence of cations. These cations were not present in the reverse osmosis purified deionised water used in the media preparation trials or the distilled water used in the water solubility test, hence higher dissolved test material concentrations were obtained in the diluents.
Based on the results of the recovery analyses conducted, it was considered appropriate to base the concentrations employed in the range-finding test on a nominal saturated solution concentration of 11 mg/L. A series of dilutions were made from this saturated solution to give further stock solutions of 1.1, 0.11 and 0.011 mg/L. An aliquot (500 ml) of each 0.11, 0.11, 1.1 and 11 mg/L stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 0.011, 0.11, 1.1 and 11 mg/L.
The control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test a sample of each test and control cultures was removed and the cell density determined using a Coulter Multisizer II Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated at 24 degC +/- 1 under continuous illumination (approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined.

Definitive test:
Based on the results of the range-finding test and the preliminary recovery analyses conducted, the test material solutions for the definitive test were prepared by shaking an excess (150 mg/L) of test material in culture medium at approximately 300 rpm at a temperature of 30 degC for a period of 48 hours. After 48 hours shaking was stopped and any undissolved test material was removed by filtration (0.2 micron) through a pre-conditioned filter to give a saturated solution of the test material at a nominal concentration of 11 mg/L from which dilutions were made to produce the remaining test groups of 5.5, 2.75, 1.38 and 0.69 mg/L.

Exposure Conditions:
As in the range-finding test 250 ml glass conical flasks were used. Three flasks each containing 100 ml of solution were prepared for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.17E6 cells per ml. Inoculation of 500 mL of test medium with 2.5 mL of this algal suspension gave an initial cell density of 10E4 cells per ml and had no significant dilution effect.
The flasks were plugged with polyurethane foam bungs and incubated at 24 =/- 1 degC under continuous illuminations (approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 5.8 - 7.5 mg/l.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

From the data it is clear that both the growth (r) and biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72 hour exposure period.
The percentage inhibition values (Ia) were plotted against test concentration a line fitted by eye and the EC50 value with respect to the area under the growth curve, EbC50 (72 h) read from the graph.
Percentage reductions in growth rate and the EC50 value with respect to growth rate, ErC50 value (0 - 72 h) were calculated.
Accordingly the following results were determined from the data:
EbC50 (72 h): 6.6 mg/L; 95% confidence limits 5.8-7.5 mg/L
ErC50 (0 - 72 h): 10 mg/L
Where EbC50 is the test concentration that reduced biomass by 50% and ErC50 is the test concentration that reduced specific growth rate by 50%.
The 95% confidence limits were calculated using the method of Litchfield and Wilcoxon 1949.
Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.69 mg/l test concentration (P<0.05) and, therefore the No Observed Effect Concentration (NOEC) was 0.69 mg/L.
The following data show that the cell concentration of the control cultures increased by a factor of 66 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:
Mean cell density of control at 0 hours: 9.34E3 cells per mL
Mean cell density of control at 72 hours: 6.13E5 cells per mL

At the start of the test all control and test cultures were observed to clear colorless solutions. After the 72 hours test period all control, 0.69, 1.38 and 2.75 mg/L test cultures were observed to be green dispersions. The 5.5 mg/L test cultures were observed to be pale green dispersions whilst the 11 mg/L test cultures were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The effect of ZMB2 on the growth of Scendesmus subspicatus has been investigated over a 72 hour period and based on the measured test concentrations gave an EbC50 (72 h) value of 6.6 mg/L; 95% confidence limits 5.8 - 7.5 mg/L and an Er50 (72 h) of 10mg/L (it was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits). The No Observed Effect Concentration at 72 hours was 0.69 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

36.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Validity criteria fulfilled:

not specified

Conclusions:

The growth rate of algae was determined as ErC50 (0-72 h) = 62.24 mg/L and an ErC10 (0-72 h) = 36.1 mg/L. Furthermore, a NOEC of 25.0 mg/L and a LOEC of 50.0 mg/L was calculated.

Executive summary:

In a one-to-one read-across approach, the substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (source substance) is considered appropriate for direct read-across (one-to-one) to zinc 4-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide 7-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide (target substance) for the endpoint toxicity to aquatic algae and cyanobacteria. In conclusion, the test item 72 h EC50, EC10 and NOEC for growth rate inhibtion to algae were 62.2 mg/L (95 % confidence limit: 61.5 -63 mg/L), 36.1 (95 % confidence limit: 35.2 -36.9 mg/L) and 25 mg/L, respectively. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Toxicity estimated using ECOSAR class (Thioureas) and Neutral Organic SAR (Baseline Toxicity).

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Guideline:

other: REACH guidance on QSARs R.6, May/June 2008

Principles of method if other than guideline:

Toxicity estimated using ECOSAR class (Thioureas) and Neutral Organic SAR (Baseline Toxicity).

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

0.212 mg/L

Remarks on result:

other: ECOSAR Class (Thioureas)

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

7.301 mg/L

Remarks on result:

other: Neutral Organic SAR (Baseline Toxicity)

Details on results:

Thioureas :

For Fish and Daphnid Acute Toxicity Values: If the log Kow of the chemical is greater than 5.0, or if the compound is solid and the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

For Green Algae Acute Toxicity Values: If the log Kow of the chemical is greater than 6.4, or if the compound is solid and the EC50 exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

For All Chronic Toxicity Values: If the log Kow of the chemical is greater than 8.0, or if the compound is solid and the ChV exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

Validity criteria fulfilled:

yes

Remarks:

QSAR validated for substance type (organosulphur).

Conclusions:

1,3-dihydro-4(or5)-methyl-2H-benimidazole-2-thione, zinc salt was estimated to have a Chronic Value (ChV) to green algae of 0.212 mg/l using the ECOSAR Class (Thioureas) method and a ChV to green algae of 7.301 mg/l using the Neutral Organic SAR (Baseline Toxicity) method.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2013

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

0.155 mg/L

Remarks on result:

other: ECOSAR Class (Thioureas)

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

11.436 mg/L

Remarks on result:

other: Neutral Organic SAR (Baseline Toxicity)

Validity criteria fulfilled:

yes

Remarks:

QSAR validated for substance type (organosulphur).

Conclusions:

The test item was estimated to have a Chronic Value (ChV) to green algae of 0.155 mg/l using the ECOSAR Class (Thioureas) method and a ChV to green algae of 11.436 mg/l using the Neutral Organic SAR (Baseline Toxicity) method.

Executive summary:

In a one-to-one read-across approach, the substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (source substance) is considered appropriate for direct read-across (one-to-one) to zinc 4-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide 7-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide (target substance) for the endpoint long-term toxicity to fish. In conclusion, the test item was estimated to have a Chronic Value (ChV) to green algae of 0.155 mg/l using the ECOSAR Class (Thioureas) method and a ChV to green algae of 11.436 mg/l using the Neutral Organic SAR (Baseline Toxicity) method. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Toxicity estimated using ECOSAR class (Thioureas) and Neutral Organic SAR (Baseline Toxicity).

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Guideline:

other: REACH guidance on QSARs R.6, May/June 2008

Principles of method if other than guideline:

Toxicity estimated using ECOSAR class (Thioureas) and Neutral Organic SAR (Baseline Toxicity).

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

0.155 mg/L

Remarks on result:

other: ECOSAR Class (Thioureas)

Dose descriptor:

other: Chronic Value (ChV)

Effect conc.:

11.436 mg/L

Remarks on result:

other: Neutral Organic SAR (Baseline Toxicity)

Details on results:

Thioureas :

For Fish and Daphnid Acute Toxicity Values: If the log Kow of the chemical is greater than 5.0, or if the compound is solid and the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

For Green Algae Acute Toxicity Values: If the log Kow of the chemical is greater than 6.4, or if the compound is solid and the EC50 exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

For All Chronic Toxicity Values: If the log Kow of the chemical is greater than 8.0, or if the compound is solid and the ChV exceeds the water solubility by 10X, no effects at saturation are predicted for these endpoints.

Validity criteria fulfilled:

yes

Remarks:

QSAR validated for substance type (organosulphur).

Conclusions:

1,3-dihydro-5-methyl-2H-benzimidazole-2-thione was estimated to have a Chronic Value (ChV) to green algae of 0.155 mg/l using the ECOSAR Class (Thioureas) method and a ChV to green algae of 11.436 mg/l using the Neutral Organic SAR (Baseline Toxicity) method.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-06-21 till 2011-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling Scheduel: Concentration of 100 mg/L was measured at 0 and 72 h; and concentration of 0 mg/L at 72 h only.
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Vehicle:

no

Details on test solutions:

125.2 mg of the test item were added to 1 litre of dilution water and treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer.
Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 µm and an aseptic filter (Sartobran 150) with a pore size of 0.45 + 0.2 µm. The pH was measured to be 7.9.
The concentration of the test item was determined analytically by HPLC to be 106.345 mg/L. And nominal concentrations of 6.3, 12.5, 25, 50 and
100 mg/L.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 2.63 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

1.3 °dH, 22.5 mg/L CaCO3

Test temperature:

21 - 24 °C

pH:

Control: 7.9 at 0 h and 9.2 at 72 h
100 mg/L: 7.9 at 0 h and 8.1 at 72 h
Test item concentration (6.3, 12.5, 25 and 50 mg/L): 7.9 at 0 h and 8.3 - 10.2 at 72 h
The ph values in the test item slightly increased by more than 1.5 pH units. This increase is not regarded to be relevant to the results as all validity criteria were met.

Nominal and measured concentrations:

6.3, 12.5, 25, 50 and 100 mg/L (nominal) plus control
Effective concentrations ranged from 97.4 % to 101.8 % of nominal values at 0 hours, and from 97.0 % to 101.3 % of nominal values at 72 hours.

Details on test conditions:

- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers test volume: 100 mL
- Culturing apparatus: Light chamber in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks.
- Light intensity: A light intensity ranging from 60 to 120 µE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 5 test concentrations plus 1 control
3 replicates per concentration, 3 replicates per control
Initial cell density in the test cultures approximately 5000 cells per millilitre.
- Test item concentration/s: 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration: stock solution
- Duration of exposure: 72 hours
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

62.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 61.5 – 63.0

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

36.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 35.2 – 36.9

Duration:

72 h

Dose descriptor:

other: NOEC [r]

Effect conc.:

25 mg/L

Duration:

72 h

Dose descriptor:

other: LOEC [r]

Effect conc.:

50 mg/L

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

41.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 39.9 – 42.3

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

30.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 28.3 – 32.5

Duration:

72 h

Dose descriptor:

other: NOEC [y]

Effect conc.:

25 mg/L

Duration:

72 h

Dose descriptor:

other: LOEC [y]

Effect conc.:

50 mg/L

Validity criteria fulfilled:

yes

Remarks:

(The factor of the biomass parameter was 170, thus > 16; The mean of the replcate coefficients of variation was 10.5 %, thus < 35%; The coefficient of variation of the mean specific growth rate replicates in the control was 0.7 %, thus < 7%.)

Conclusions:

The growth rate of algae was determined as ErC50 (0-72 h) = 62.24 mg/L. Furthermore, a NOEC of 25.0 mg/L and a LOEC of 50.0 mg/L was calculated.

Executive summary:

In order to test acute toxicity to algae of 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, Desmodesmus subspicatus was exposed to the test solution of five nominal concentrations of the test substance ( 6.3, 12.5, 25, 50 and 100 mg/L) and blank control solution for a period of 72 hours under static conditions. The cell densities were measured at 24 hours intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. The measured concentrations confirm that deviation from the nominal concentrations was less than 30 % ( measured concentrations were in the range of 97.0 - 101.8 % of nominal concentrations). The growth rate of algae was determined as ErC50 (0-72 h) = 62.24 mg/L and a NOEC of 25.0 mg/L and a LOEC were of 50.0 mg/L were calculated. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

Description of key information

Key Study:

Toxicity to aquatic algae and cyanobacteria (Scendesmus subspicatus) 72 hour ErC50 = 10 mg/L and NOErC 0.69 mg/L; OECD 201; Safepharm (2003).

Supporting studies:

Toxicity to aquatic algae and cyanobacteria (Scendesmus subspicatus) 72 hour EC50 = 62.2 mg/L and NOErC = 25 mg/L; OECD 201; Currenta (2011).

Toxicity to aquatic algae and cyanobacteria (QSAR) Chronic toxicity value (ChV) = 0.212 mg/L; ECOSAR v1.0; ECOSAR (2013)

Toxicity to aquatic algae and cyanobacteria (QSAR) ChV = 0.155 mg/L; ECOSAR v1.0; ECOSAR (2013) (data from read-across substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, used in a one-to-one approach)

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

0.69 mg/L

Additional information

Key study:

Safepharm, 2003- In accordance with OECD Guideline 201 (Alga, Growth Inhibition Test) the effect of ZMB2 on the growth of Scendesmus subspicatus has been investigated over a 72 hour period. The algae was exposed to five concentrations of ZMB2, 0.69, 1.38, 2.75, 5.5 and 11 mg/L. The concentrations were made from a serially diluted stock solution. The stock solution was prepared by adding 75 mg of test item to 500 mL of culture medium, this was stirred for 48 hours and then filtered through a 0.2 µm mesh to remove particulates. The validity criteria were met.

The EbC50 (72 h) (measured) result is 6.6 mg/L; 95% confidence limits 5.8 - 7.5 mg/L and an Er50 (72 h) (measured) result is 10 mg/L. It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits. The 72 hour No Observed Effect Concentration (NOEC) is 0.69 mg/L (Safepharm, 2003).

Supporting studies:

ECOSAR, 2013 -Supporting data were generated using the United States (US) Environmental Protection Agency (EPA) Ecological Structure Activity Relationships (ECOSAR) v1.0 tool. ECOSAR is validated for organosulphates, a chemical class which includes the read-across substance MB2 (CAS Number 27231-36-3), and is structurally similar to ZMB2 (CAS Number 61617-00-3).

ZMB2 was estimated to have a Chronic Value (ChV) to algae of 0.212 mg/L using the ECOSAR Class (Thioureas) method (ECOSAR, 2013). This result shows good concordance with the derived NOEC from the experimental study conducted on ZMB2 (Safepharm, 2011).

The read across substance, MB2, was estimated to have a Chronic Value (ChV) to algae of 0.155 mg/L using the ECOSAR Class (Thioureas) method (ECOSAR, 2013). The result is conservartive in comparison with experimental data (Currenta, 2011)

Currenta, 2011- In order to test acute toxicity to algae of MB2, Desmodesmus subspicatus was exposed to the test solution of five nominal concentrations of the test substance ( 6.3, 12.5, 25, 50 and 100 mg/L) and blank control solution for a period of 72 hours under static conditions. The concentrations were made by the serial diltuion of a stock solution. This was produced by adding 125.2 mg of MB2 to 1 litre of test medium. The mixture was placed in an ultrasonic bath for 1 hour, after this preiod the substance was stirred for 24 hours and then filtered through a 0.2 µm mesh to remove partiuclates and leave only the soluble fraction.

The cell densities were measured at 24 hours intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. The measured concentrations confirm that deviation from the nominal concentrations was less than 30 % ( measured concentrations were in the range of 97.0 - 101.8 % of nominal concentrations). The growth rate of algae was determined as ErC50 (0-72 h) = 62.24 mg/L. Furthermore, a NOEC of 25.0 mg/L and a LOEC of 50.0 mg/L was calculated. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study. The study shows that the ECOSAR estimate is conservative.

Discussion:

As data are available on the target substance and the appropriate methodology was used (Safepharm 2003), this will be conisdered during the hazard assessment of ZMB2. The ErC50 will be used as an acute value and the NOEC will be used to fulfill the long-term toxicity to aqautic algae and cyanobacteria in accordance with ECHA guidance (ECHA Guidance on information requirements and chemical safety assessment Chapter R.10: Characterisation of dose[concntration]-response for the environment, 2008).